GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF), an immunomodulator of hematopoietic cells, has also been shown to stimulate human keratinocyte proliferation in vitro and speed healing of wounds in the skin of lepromatous leprosy patients. In this study we have examined the in vivo effects of recombinant human GM-CSF on epidermal keratinocyte proliferation and on expression of proteins marking regenerative epidermal growth. Skin biopsies from GM-CSF injected cutaneous sites were obtained between 1 and 6 d following administration of 7.5 or 15 micrograms of the growth factor. Activation of keratinocyte proliferation, quantified as the expression of the Ki67+ nuclear antigen, was noted 1 d following GM-CSF administration. A regenerative epidermal phenotype, demonstrated by immunohistochemical staining of cellular proteins involucrin, filaggrin, and keratin 16, was similarly noted as early as 1 d following GM-CSF injection. This phenotype persisted as late as 6 d post-injection. These results suggest that GM-CSF injection into human skin induces keratinocyte proliferation as well as regenerative differentiation of the epidermis. To date no other cytokine has been shown to be mitogenic for human keratinocytes both in vivo and in vitro or to alter keratinocyte differentiation along the "alternate" or regenerative pathway.

Evaluation of polymerase chain reaction amplification of Mycobacterium leprae-specific repetitive sequence in biopsy specimens from leprosy patients.

Biopsy specimens were obtained from 102 leprosy patients before chemotherapy and examined by polymerase chain reaction (PCR) using the primers amplifying the 372-bp DNA of a repetitive sequence of Mycobacterium leprae. The PCR results were then compared with bacterial indices (BI) of slit-skin smears and biopsy specimens. The intensities of DNA bands were in general correlated with the numbers of acid-fast bacilli, and even a sample with only one organism gave a PCR positive result. Ten 5-micron sections from each frozen tissue sample were pooled and processed for DNA preparation. PCR was positive for 11 (73.3%) of 15 biopsy specimens with BI of 0 determined for the paraffin sections from the same biopsy samples. PCR also gave positive results for 84 (96.6%) of 87 BI positive biopsy samples. Although the difference in overall results between the two methods was not statistically significant, PCR seemed to have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli. Further evaluation of PCR using more specimens from leprosy patients who are bacteriologically negative is warranted to ensure PCR's advantage over the conventional microscopic examination for the diagnosis of leprosy.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

Serological monitoring of previously treated lepromatous patients during a course of multiple immunotherapy treatments with heat-killed Mycobacterium leprae and BCG.

Two-hundred and seventy lepromatous patients who had completed treatment received multiple treatments with heat-killed M. leprae and BCG and were monitored for changes in humoral responses to M. leprae-specific antigens. These patients were divided into four treatment groups: placebo (n = 69); BCG (n = 68); M. leprae only (n = 71); and BCG + M. leprae (n = 62). They were monitored for 15 months, receiving five inoculations for each treatment regimen. Two ELISA systems, one measuring antibodies to M. leprae-specific epitopes of the phenolic glycolipid I (NDO-ELISA) and the other of 36-kD protein antigens (INH-ELISA) were used to measure serological changes during this period of immunotherapy. We found no significant increase in serological reactivity with the different treatments, as measured by NDO-ELISA. INH-ELISA similarly showed no significant changes, with the exception of increased values in a small group 13% (36/270) which became skin test-positive during the course of the study. The NDO-ELISA results indicate that use of heat-killed M. leprae or BCG + heat-killed M. leprae did not stimulate the humoral response to the semi-synthetic PG-I antigens of M. leprae. Thus, the NDO-ELISA may be useful in monitoring the outcome of vaccine trials in which killed M. leprae or M. leprae fractions are used, since seroconversion may indicate disease, rather than a response to the vaccine material.

Influence of Mycobacterium leprae and its soluble products on the cutaneous responsiveness of leprosy patients to antigen and recombinant interleukin 2.

Experiments were carried out in the skin of patients with leprosy to examine whether suppressor cell populations either exist in the skin of multibacillary lepromatous leprosy patients, can be activated with antigen, or are induced to emigrate into a cutaneous site from the circulation. For this purpose, purified protein derivative of tuberculin, a delayed-type antigen that generates a cell-mediated immune response, was introduced into the skin alone or with nonviable Mycobacterium leprae bacilli. Areas of induration and the resulting numbers and phenotypes of emigratory cells were not influenced by M. leprae and its products. Further studies examined the ability of M. leprae and its soluble products to modify the cutaneous response to intradermal injection of recombinant interleukin 2 (IL-2), a lymphokine that mimics a cell-mediated response. Neither the simultaneous injection of M. leprae and IL-2, nor the prior injection of M. leprae followed in 2 days by IL-2, nor the prior administration of IL-2 followed in 4 days by M. leprae, into the same skin site, modified the zone of induration generated by IL-2. In addition, the immunocytochemical and histopathological evaluation of biopsy specimens of skin sites showed no difference between sites injected with IL-2 and sites injected with IL-2 and M. leprae. We conclude that suppressor T cells, if they exist, do not influence the gross or microscopic responsiveness of a cell-mediated skin reaction to antigen and IL-2. IL-2 did, however, enhance the responsiveness of skin-test-positive tuberculoid patients and family contacts to M. leprae antigens by a synergistic effect on the zone of induration and local cell accumulation.